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BOR - Papers in Press, published online ahead of print January 17, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.054734
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BIOLOGY OF REPRODUCTION 76, 749–758 (2007)
DOI: 10.1095/biolreprod.106.054734
© 2007 by the Society for the Study of Reproduction, Inc.

Expression Pattern of Prokineticin 1 and Its Receptors in Bovine Ovaries During the Estrous Cycle: Involvement in Corpus Luteum Regression and Follicular Atresia

Tatiana Kisliouk 2, Aharon Friedman 3, Eyal Klipper 2, Qun-Yong Zhou 4, Dieter Schams 5, Nadia Alfaidy 6, and Rina Meidan 1 2

Department of Pharmacology,4 University of California, Irvine, California 92697 Institute of Physiology,5 Technical University of Munich, D-85350 Freising, Germany INSERM EMI 105,6 Départment des Sciences du Vivant sud, Commissariat à l'Energie Atomique de Grenoble, 38000 Grenoble, France Sections of Reproduction2 and Immunology,3 Department of Animal Sciences, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

ABSTRACT

Prokineticin 1 (PROK1), also termed endocrine gland-derived vascular endothelial growth factor (endocrine gland-derived VEGF), is a newly identified protein assigned with diverse biologic functions. It binds two homologous G protein-coupled receptors, PROKR1 and PROKR2. To better understand the roles of PROK1 and its receptors in ovarian function, their expression was determined in follicles and corpora lutea (CLs) at different developmental stages. PROK1 mRNA levels were low at early luteal stage and midluteal stage, but increased sharply during natural or induced luteolysis. High PROK1 mRNA levels also were found in atretic follicles. This profile of PROK1 expression was opposite to that of the well-established angiogenic factor VEGF. Of the two receptor-type expressions, PROKR1 but not PROKR2 was correlated positively with its ligand. Immunohistochemical staining revealed that PROK1 was located mainly within the muscular layer of arterioles, and during regression it also was localized to macrophages and steroidogenic cells. The expression pattern of ITGB2 mRNA, a leukocyte cell marker, overlapped that of PROK1, thus suggesting that leukocyte infiltration may explain the elevated expression of PROK1 in atretic follicles and regressing CL. Indeed, flow cytometry analyses showed that nearly all beta-2 integrin chain (ITGB2)-positive cells also were stained with anti-PROK1 and that significantly more ITGB2/PROK1 double-stained cells were present in degenerating follicles and CL. Furthermore, when challenged in vitro with PROK1, adherent, mononuclear cell numbers and TNF levels were elevated, indicating that PROK1 triggers monocyte activation. Together, these data suggest that PROK1, acting via PROKR1, may be involved in the recruitment of monocytes to regressing CL and atretic follicles and their consequent activation therein.

apoptosis, corpus luteum, corpus luteum function, cytokines, follicle, follicular atresia, inflammation, luteolysis, macrophages, ovary

INTRODUCTION

Prokineticin 1 (PROK1) is a newly identified protein [1, 2]. Together with its close homolog, PROK2, these secreted proteins belong to a family of structurally related peptides consisting of 80–90 amino acids, including 10 conserved cysteines, which create a five-disulphide bridged motif and identical amino termini, AVIT [1, 3]. The sequence of the first four identical residues, AVIT, is used as an epithet for this protein family [3]. PROK1 is expressed in various tissues (e.g., in steroidogenic glands, along the gastrointestinal tract, and in the nervous system) [1, 2, 4]. Recently, PROK1 also was found in immune cells and in inflamed human tissues [5, 6]. Interestingly, while PROK1 mRNA was identified in the ovaries of different species [2, 7–9], PROK2 mRNA, which is highly expressed in the testis [10], was almost undetectable in the ovary [7–9].

PROK1 is the cognate ligand for two closely homologous G protein-coupled receptors termed PROK receptors (PROKR1 and PROKR2); these receptors bind both PROK1 and PROK2 with almost equal affinity [11–13]. Similarly to the peptides, PROKRs are widely distributed: PROKR2 is more restricted to the central nervous system, whereas receptor subtype 1 is somewhat more abundant in peripheral tissues [4, 11–14]. We found that PROKR1 also is the common receptor form in the bovine ovary: follicular and luteal steroidogenic cells (SCs) predominantly express PROKR1, whereas corpus luteum (CL)-derived endothelial cells (ECs) express, in addition to PROKR1, the other subtype, PROKR2 [15]. We also reported that in luteal ECs the two receptor types were inversely regulated in response to stress conditions [9].

PROKs are multifunctional proteins; the list of their biologic activities is growing rapidly [1, 2, 4–6, 16–18]. They influence circadian rhythms [4] and are involved in neuronal survival [16], angiogenesis [2, 9, 10], and hematopoiesis, including hematopoietic cell mobilization [5]. More recently, PROKs were shown to alter monocyte differentiation and function [6, 18]. Studies in Prokr1 knockout mice indicated that all the activities exerted by PROK2 on macrophages were mediated by PROKR1 [18]. Recently, we demonstrated an important paracrine role for PROK1 in luteal function: it promoted proliferation and survival of bovine luteal ECs [9]. Acting via PROKR1 present in luteal SCs, PROK1 also increased VEGF mRNA expression, implying that it also could affect luteal angiogenesis indirectly, via vascular endothelial growth factor (VEGF) [9].

Dynamic changes in vasculature and cell populations occur during the life span of the CL [19–24]. While luteal development is accompanied by a dramatic increase in the number of blood vessels, luteal demise is characterized by increased chemokine expression and leukocyte infiltration [23, 25–28]. PROK1 may therefore assume different roles at different stages of the cycle.

To better understand the roles of PROK1 and its two receptors in ovaries, the profile of their gene expression was determined throughout the bovine estrous cycle. To localize PROK1, follicles (healthy vs. atretic) and CL (mid vs. regressed) were labeled with anti-PROK1 antisera for immunohistochemistry or were sorted by a flow cytometer. As PROK1 was found to be upregulated in atretic follicles and regressing CL, two niches that have been shown to harbor inflammatory macrophages, we studied PROK1 expression and effects during monocyte activation.

MATERIALS AND METHODS

Materials

Cell media: M-199, Dulbecco modified Eagle medium-F12 (DMEM-F12; 1:1), RPMI-1647, Hanks balanced salt solution (HBSS), L-glutamine, gentamycin sulphate, and XTT proliferation kit were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). Collagenase I, collagenase IV, hyaluronidase, and DNase I were from Worthington Biochemical Corp. (Freehold, NJ). Recombinant human sFas ligand (FASLG) was obtained from Cytolab Ltd. (Rehovot, Israel). Bovine serum albumin (BSA), donkey serum, N-acetyl-D-sphigosine (C2-ceramide), and Histopaque-1077 were purchased from Sigma (St. Louis, MO). TRI Reagent was from MRC (Cincinnati, OH). Fetal bovine serum (FBS), SuperScript II RNase H- Reverse Transcriptase, and random hexamer oligodeoxynucleotides were purchased from Invitrogen Corp. (Paisley, Scotland). Deoxynucleotide triphosphates (dNTPs) were from Bioline GmbH (Luckenwalde, Germany). Oligo-dT and oligonucleotide primers were synthesized by Sigma-Genosys (Rehovot, Israel). The real-time PCR SYBR Green Master Mix Kit was from Eurogentec (Seraing, Belgium). Progesterone and estradiol radioimmunoassay kits were purchased from DPC (Los Angeles, CA). The rabbit serum containing polyclonal PROK1 antibody raised against the human PROK1 peptide (residues 84–96: LLCSRFPDGRYRC) was from Covalab (Lyon, France) [29]. The beta-2 integrin chain (ITGB2, also known as CD18) monoclonal antibody was obtained from VMRD (Pullman, WA). Cy3-conjugated donkey anti-rabbit IgG (H+L) and fluorescein (FITC)-conjugated F(ab')2 fragment donkey anti-mouse IgG (H+L) were from Jackson ImmunoResearch Laboratories (West Grove, PA). Antiserum raised against cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A) was the generous gift of Dr. S. Silavin (Adeza Biomedical, Sunnyvale, CA). DAKO anti-rabbit EnVision HRP labeled polymer and 3-amino-9-ethylcarbazole chromogen (AEC) were obtained from DakoCytomation (Carpinteria, CA). Strain of Escherichia coli O2:K1 was provided by Dr. E.D. Heler (Hebrew University of Jerusalem, Rehovot, Israel). O2:K1 bacteria were grown in liquid broth, then harvested by centrifugation, resuspended in buffer consisting of 20 mM Tris and 5 mM EDTA (pH 8), and sonicated on ice for three periods of 3 min each. Sonicated E. coli extract was centrifuged at 3000 x g for 20 min and passed through a 0.22-µm filter to remove insoluble material. Recombinant human PROKs (PROK1 and PROK2) were produced and purified as described previously [1].

Collection of CLs

CLs were collected at a local slaughterhouse, and luteal stage was determined by macroscopic examination of the ovaries according to criteria described by M. Fields and P. Fields [30]. CLs were divided into four groups: early (Days 3–5), mid (Days 8–12), late (Days 15–18), and regressing (after Day 18). For timed CL regression, cows at the midluteal phase (Days 8–12) were injected with prostaglandin F2{alpha} (PGF2{alpha}) analog cloprostenol (Estrumate, Intervet, Germany), and samples of CL were collected at 24, 48, 64, and 72 h thereafter [31]. The experimental protocol was approved by the Institutional Care and Use Committee of the Technical University of Munich. All CLs were immediately frozen in liquid nitrogen and stored at –80°C until RNA extraction.

Cell Isolation and Culture

Luteal cells. CLs at the midluteal phase were dispersed by means of collagenase IV as previously described [9]. Briefly, CLs were sliced, washed, and dispersed in M-199 containing 0.5% BSA and collagenase IV (420 U/ml). Dispersed luteal cells were seeded and cultured overnight in DMEM-F12 containing 10% FBS, 2 mM L-glutamine, and 50 (g/ml gentamicin sulfate. In experiments aimed at examining the effects of FASLG and C2-ceramide on cell viability, 10 000 cells/well were seeded in 96-well culture dishes, and for determination of PROK1 mRNA expression, 0.5 x 106 to 1 x 106 cells/well were seeded in 6-well plates. The treatments, FASLG (100 ng/ml) and C2-ceramide (20 µM), were added in DMEM-F12 including 0.5% BSA for 24 h.

Peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMCs) were collected from the jugular venous blood of prepubertal heifers. Heparinized whole blood was diluted 1:2 with HBSS. Then, 35 ml diluted blood solution was layered on 15 ml Histopaque-1077 and centrifuged for 30 min at 950 x g at room temperature. The ring of mononuclear cells was carefully aspirated and was resuspended in 20 ml HBSS, with subsequent centrifugation of the cells at 260 x g for 10 min. The cells were washed three times in HBSS, resuspended in medium RPMI with 10% FBS, and then seeded at 10 x 106 to 15 x 106 cells/well and 0.1 x 106 to 0.2 x 106 cells/well in 6-well and 96-well plates, respectively. PBMCs were incubated in the presence or absence of E. coli sonicate (15 µg/ml) or PROKs (50 nM) for up to 48 h. Nonadherent cells (mainly lymphocytes) were removed from the adherent monocyte layer by washing with HBSS.

Coculture of luteal cells with PBMCs. Dispersed luteal cells (as detailed above) were seeded at 0.5 x 106 cells/well in six-well plates and cultured overnight in DMEM-F12 with 10% FBS, and then medium was changed to RPMI containing 10% FBS. PBMCs were added to luteal cells at 10 x 106 to 15 x 106 cells/well and were incubated with PROKs (50 nM) for up to 48 h.

Granulosa and theca cells. Ovaries with large follicles (>10 mm in diameter) were collected at a local slaughterhouse. To determine the status of the follicles, estradiol and progesterone concentrations were measured in follicular fluid by DPC estradiol and progesterone kits according to the manufacturer's instructions. Follicles with estradiol concentration in follicular fluids >100 ng/ml and a ratio of estradiol:progesterone >1 were classified as healthy large follicles [32–34]. Granulosa cells (GCs) were enzymatically dispersed as previously described [35]. Briefly, GCs were aspirated with DMEM-F12 containing 0.1% hyaluronidase, 0.1% collagenase I, and 5 µg/ml DNase I. The theca cell (TC) layer was peeled from the ovary with fine forceps and incubated in 0.25% trypsin/0.02% EDTA at 37°C for 15 min, followed by 45 min of incubation in DMEM with 3% collagenase I and 10 µg/ml DNase I. Then, RNA was isolated, reverse transcribed, and subjected to SYBR Green real-time PCR for determination of aromatase (CYP19A1) and VEGF mRNA expression as detailed below. GCs of healthy follicles expressed more than 100 arbitrary units of CYP19A1 and 4 arbitrary units of VEGF.

Fluorescent-Activated Cell Sorting Analyses

Dispersed cells from the GC layers of healthy and atretic follicles as well as dispersed luteal cells from midcycle and regressed stages were fixed with 2% paraformaldehyde in PBS for 30 min at 4°C, permeabilized in 0.5% Triton X-100 for 15 min at 37°C, washed in fluorescent-activated cell sorting (FACS) buffer (0.5% BSA and 0.005% Na3N in PBS), and incubated with 2% donkey serum (to block nonspecific binding) for 1 h at room temperature. Cells were incubated sequentially with the following antibodies: 1) anti-PROK1 (1:50; 18 h at 4°C), 2) Cy3-conjugated donkey anti-rabbit IgG (1:300; 40 min at 4°C), 3) anti-ITGB2 (1:100; 40 min at 4°C), and 4) FITC-conjugated F(ab')2 fragment donkey anti-mouse IgG (1:150; 40 min at 4°C). The cells (10 000 to 30 000) then were resuspended in FACS buffer and analyzed for both FITC and Cy3 fluorescence on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Cells that had been incubated only with Cy3-conjugated donkey anti-rabbit IgG and FITC-conjugated F(ab')2 fragment donkey anti-mouse IgG served as a negative control.

RNA Isolation and Real-Time PCR

Total RNA was isolated from the cells with TRI Reagent according to the manufacturer's instructions. PCR reactions were performed using PE Biosystems' GeneAmp 5700 sequence detection system, with the SYBR Green I PCR kit used as previously described [9]. Briefly, each real-time reaction (18 µl) contained SYBR Green Master Mix that comprised: ROX passive reference, 200 µM dNTPs, including deoxyuridine triphosphate; 5 mM MgCl2; uracil N-glycosylase and Amplitaq HotGoldStar DNA polymerase; 0.54 µl of a 1:10 000 dilution of SYBR Green stock solution; 1.5 mM dNTPs; 10 nM of each primer; and 5–50 ng cDNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as a standard for mRNA expression in CL and GCs of follicles. Beta-actin (ACTB) was used as a standard for mRNA expression by cultured cells. Dissociation curve analysis was run following each real-time experiment to confirm the presence of only one product and the absence of formation of primer dimmers. The threshold cycle number (CT) for each tested gene X was used to quantify the relative abundance of the gene: 2–(Ct gene X-CtG3PDH) x 1000. Table 1 presents a list of primers.


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  		TABLE 1. PCR primers used in this study.

Immunohistochemistry

CL and follicles were fixed in 4% (v/v) paraformaldehyde, embedded in paraffin, and cut into 5-µm sections. The sections were dewaxed in xylene and rehydrated using decreasing ethanol concentrations. Endogenous peroxidase activity was quenched by pretreatment with 3% (v/v) hydrogen peroxide in methanol for 30 min. Antigen retrieval was performed by treating sections for 5 min in a microwave in boiling citrate buffer (Na citrate 10 mM, pH 6.0). Tissue sections then were washed in PBS and incubated for 1 h with normal horse serum (10%) that served as a blocking agent for nonspecific binding. The serum containing polyclonal PROK1 or CYP11A rabbit antibodies (dilution 1:50 in blocking solution for PROK1 and 1:100 for CYP11A) was added for 18 h. After three washes in PBS, the slides were incubated with DAKO anti-rabbit EnVision HRP labeled polymer for 30 min. After a final PBS wash, the immunoreactive proteins were visualized with AEC solution and then counterstained with hematoxylin.

Evaluation of Cell Number

The number of cells in culture wells was determined by the XTT assay, which measures the conversion of a tetrazolium salt (XTT) by metabolically active, viable cells. For determination of luteal cell numbers, 10 000 cells/well were seeded in 96-well culture dishes and incubated for 24 h as indicted above, then medium was replaced to basal DMEM-F12 (100 µl). PBMCs (0.1 x 106 to 0.2 x 106 cells/well) were seeded in 96-well plates and incubated as described above. Nonadherent cells (mainly lymphocytes) were removed from the adherent monocyte layer by washings with HBSS. XTT (50 µl) was added to cells containing 100 µl basal RPMI media. The cells then were incubated for 4–7 h at 37°C. The absorbance was read at 450 nm. A blank well reading was subtracted from each value, and cell viability was determined by dividing the mean of treated wells by the mean of control (n = 3 for each treatment or control). The percentage of viable adherent cells was calculated as follows: cell viability (%) = 100 x (treatment)/(control).

Statistical Analysis

Data are presented as means ± SEM. The one-way ANOVA Tukey-Kramer test was used to determine the statistical difference between different stages of luteal phase and to evaluate the effect of PROKs on monocyte activation. Student t-test was used to determine the statistical difference between gene expressions in healthy versus atretic follicles, to compare ITGB2 expression in follicles and CL, to evaluate the effects of FASLG and C2-ceramide on cell viability and the mRNA expression of PROK1 by luteal SCs and, finally, to analyze the effect of monocyte activation on PROK1 mRNA expression and the effects of PROKs on TNF mRNA expression. Correlation between PROK1 and ITGB2 mRNAs within each individual GC/CL sample were examined by linear regression test. Differences were considered significant at P < 0.05.

RESULTS

Expression Profile of PROK1, VEGF, and PROK Receptor mRNA in Bovine Follicles and CL During Different Stages of the Cycle

Expression of PROK1 mRNA in CL tissue throughout the luteal phase and after spontaneous or PGF2{alpha}-induced luteolysis was determined by SYBR Green real-time PCR. Low mRNA levels of PROK1 were detected at early, mid, and late luteal stages (Fig. 1A), but were sharply elevated during luteolysis, both spontaneous (Fig. 1A) and PGF2{alpha} induced (Fig. 1B). PROK1 levels were already elevated 24 h after PGF2{alpha} and remained high at least up to 72 h after PGF2{alpha} treatment (Fig. 1B). Comparing the profiles of PROK1 and VEGF mRNA showed that these two factors were inversely expressed. As expected, VEGF mRNA levels were high in the early luteal phase and midluteal phase, reduced at the late luteal stage, and then abruptly decreased during CL regression (Fig. 1).


Figure 01
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  		FIG. 1. PROK1 and VEGF expression in the bovine CL throughout the luteal phase (A) and during PGF2{alpha}-induced luteolysis (B). RNA was isolated from CL in the early (n = 8), mid (n = 9), and late (n = 3) luteal phases; during natural regression (n = 10); and after PGF2{alpha}-induced luteolysis on Days 8–12 (n = 4 to 8 for each time point). After reverse transcription, expression of PROK1 and VEGF in each sample was determined by real-time PCR. Different letters (a, b in cases of VEGF and A, B in cases of PROK1) indicate significant differences (P < 0.05).

Similarly to PROK1, its type 1 receptor (PROKR1) mRNA increased during CL regression and reached its highest levels 72 h after PGF2{alpha}-induced luteolysis or in the naturally regressed CL (Fig. 2). Expression of PROKR2 mRNA did not significantly change throughout the cycle or during luteolysis (Fig. 2).


Figure 02
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  		FIG. 2. Expression of PROK receptors in the bovine CL at different stages of the luteal phase (A) and after PGF2{alpha}-induced luteolysis (B). The same cDNA samples as described in the Figure 1 legend were used. PROK receptor (PROKR1 and PROKR2) expression was determined by real-time PCR. Different lowercase letters indicate significant differences (P < 0.05).

The highest PROK1 mRNA levels were detected in the GCs of atretic follicles (expressing low mRNA levels of CYP19A1 and VEGF; Fig. 3A). There was an increase of nearly 20-fold in PROK1 mRNA expression in the atretic compared with the healthy follicles (P < 0.05; Fig. 3B). Similarly, the mRNA levels of subtype 1 receptor (PROKR1) also were significantly higher in atretic follicles than in their healthy counterparts (Fig. 3B). Changes in PROKR2 mRNA expression were not statistically significant (Fig. 3B). Messenger RNA levels of PROK1 and its receptors also were elevated in the TCs of atretic follicles (data not shown).


Figure 03
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  		FIG. 3. Differential expression of CYP19A1 and VEGF (A), and PROK1 and PROK receptors (B) in the GCs of healthy and atretic follicles. RNA was extracted from the GCs of large follicles (n = 25), followed by reverse transcription and real-time PCR analysis of CYP19A1 and VEGF expression. Follicles expressing high levels of CYP19A1 and VEGF mRNA (see Materials and Methods) were classified as healthy follicles (n = 15), whereas follicles with low levels of CYP19A1 and VEGF were determined to be atretic follicles (n = 10). Expressions of PROK1, PROKR1, and PROKR2 in the GCs of healthy and atretic follicles also were determined by real-time PCR. *P < 0.05, **P < 0.01 indicate a significant difference from healthy follicles.

Leukocyte numbers are elevated during follicular atresia and CL regression [23, 25, 26, 28]. To confirm this observation, we next examined expression of ITGB2, a pan-leukocyte marker, in the samples described above. As demonstrated in Figure 4, ITGB2 mRNA expression was elevated in atretic follicles (Fig. 4A) and in regressing CL (Fig. 4B). The profile of ITGB2 and PROK1 mRNA expression was similar in progression (Figs. 1 and 3). However, within each individual sample (GC or CL) there was no positive correlation between expression of ITGB2 and PROK1 mRNAs, implying that SCs, as well as infiltrating leukocytes, may be a source for PROK1.


Figure 04
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  		FIG. 4. Expression of ITGB2 mRNA in the GCs of healthy and atretic follicles (A) and CL in the early luteal phase and midluteal phase and during PGF2{alpha}-induced luteolysis (B). Messenger RNA levels were examined by real-time PCR in the GCs of healthy follicles (n = 8), GCs of atretic follicles (n = 9), and CL in the early luteal stage (n = 8), midluteal stage (n = 9), and after PGF2{alpha}-induced lyteolysis (n = 4 to 8 for each time point). In A, asterisk (*) indicates a significant difference (P < 0.05) between healthy and atretic follicles. In B, different lowercase letters indicate significant differences between stages of luteal phase (P < 0.05).

Cellular Localization of PROK1 in Follicles and CL

The cellular localization of PROK1 in follicles (healthy vs. atretic) and CL (mid vs. regressing) was determined by: 1) flow cytometry analysis of dispersed follicular (GC layer) and luteal cells, which were double stained with PROK1 and ITGB2 antibodies and 2) immunohistochemical staining of PROK1 in tissue sections prepared from follicles and CL.

As demonstrated by FACS analysis (Fig. 5), the number of ITGB2-positive cells (leukocytes) increased in atretic follicles and regressing CL (from 11% and 18% in healthy and midcycle CL to 26% and 39% in atretic follicles and regressing CL, respectively; Fig. 5B), which corresponded with the results of real-time PCR (Fig. 4). Interestingly, nearly all ITGB2-positive cells also were stained with anti-PROK1 in both follicles and CL (Fig. 5A, upper right rectangles). FACS analyses (Fig. 5B) revealed a significant increase in the number of double-stained cells in atretic follicles and regressing CL compared with healthy follicles and midcycle CL (2.3-fold and 2.1-fold, respectively). However, most follicular and luteal cells were only stained by anti-PROK1 (Fig. 5A, upper left rectangles).


Figure 05
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  		FIG. 5. FACS analyses of PROK1- and ITGB2-positive cells in the GC layer of follicles (healthy vs. atretic) and CL (midcycle vs. regressing). A) Representative flow cytometry scan of PROK1- and ITGB2-expressing cells derived from follicles and CL. B) Percentage of PROK1/ITGB2 double-stained cells in dispersed GC layer of healthy and atretic follicles and in a population of dispersed luteal cells from midcycle and regressing CL. Each column (mean ± SEM) represents the percentage of PROK1/ITGB2 double-stained cells obtained from healthy follicles (n = 10), atretic follicles (n = 9), midcycle CL (n = 4), and regressing CL (n = 3), respectively. *Significant difference (P < 0.05) between healthy and atretic follicles. +Significant difference (P < 0.05) between midcycle and regressing CL.

To specifically determine the cell types expressing PROK1, sections of follicular and luteal tissue were prepared and immunostained. PROK1 staining was detected in the GC layer of healthy and ateric follicles (Fig. 6, A and B), but a greater intensity of staining was observed in atretic follicles, where PROK1 staining also became apparent in the theca interna layer (Fig. 6B). PROK1 immunoreactivity was observed in the SCs of midcycle and regressed CL but tended to be more intense in the latter (Fig. 6, D and E). SCs were identified by staining with anti-CYP11A (Fig. 6, G and H). Macrophages present in regressed CL were strongly labeled with PROK1 antisera (Fig. 6F), and this finding is in agreement with flow cytometry data (Fig. 5). An extensive PROK1 staining also was observed in the smooth muscle cell (SMC) layer of arterioles within the midcycle and regressed CL (Fig. 6, D–F). Endothelial cells did not display PROK1 immunoreactivity in either follicles or CL (Fig. 6, B, D, and E).


Figure 06
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  		FIG. 6. Immunolocalization of PROK1 and CYP11A in bovine follicles and CL. Expression of PROK1 in the large healthy follicles (A) and atretic follicles (B). PROK1 expression in the midluteal stage (D) and regressing CL (E, C, F). CYP11A staining of luteal tissue at the midluteal phase (G) and regressing CL (H). Negative control: the midluteal CL section was incubated in the absence of antibodies for PROK1 and CYP11A (I). All sections were counterstained with hematoxylin. GC, granulosa cells; TCi, theca interna cells; TCe, theca externa cells; SC, steroidogenic cells; M, monocytes/macrophages; A, smooth muscle cells of arterioles; V, vein. Original magnification A, B, D, E, and G–I x200; C and F x400.

Since PROK1 mRNA levels were elevated in apoptotic tissues, namely in atretic follicles and regressing CL (Figs. 1 and 3B), we next examined the direct effects of factors known to induce apoptosis in ovarian tissues, such as FASLG and C2-ceramide [36, 37], on PROK1 mRNA. Treatment of luteal cells obtained from midcycle CL with FASLG (100 ng/ml, 24 h) and C2-ceramide (20 µM, 24 h) caused the death of 40% and 50% of the cells (P < 0.05), respectively, as determined by XTT assay (Fig. 7A). However, neither FASLG nor C2-ceramide significantly affected PROK1 mRNA expression (Fig. 7B).


Figure 07
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  		FIG. 7. Effects of FASLG and ceramide-2 on survival of luteal cells and PROK1 mRNA expression. Cells were grown in medium containing either 0.1% FBS alone, FASLG (100 ng/ml), or C2-ceramide (20 µM) for 24 h. A) XTT assay: the percentage of viable adherent cells was calculated as described in Materials and Methods. Data are means ± SEM from four separate experiments. B) PROK1 mRNA expression as determined by real-time PCR: within each experiment results were normalized to control (0.1% FBS alone), which received value 1. Data are means ± SEM from three independent experiments. *P < 0.05 denotes a significant difference from the control.

In Vitro Activation of PBMCs

To further study whether monocyte activation affects PROK1 gene expression, PBMCs were incubated with 15 µg/ml sonicated E. coli for 4–48 h to activate macrophage precursors. Following activation, nonadherent cells were removed, and PROK1 mRNA expression was measured in the adherent cell fraction (predominantly macrophages, as determined by light microscopy; data not shown). Stimulation of the cells with E. coli sonicate augmented PROK1 mRNA expression in a time-dependent manner: there was a 5-fold increase after 24 h (P < 0.01) of incubation, and a 7-fold increase after 48 h (P < 0.05) of incubation (Fig. 8).


Figure 08
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  		FIG. 8. Stimulation of PROK1 mRNA expression in bovine macrophages by E. coli sonicate. PBMCs were incubated with E. coli sonicate (15 µg/ml) for up to 48 h. At each indicated time point RNA was extracted from the adherent cells, and mRNA levels were determined by real-time PCR. Within each experiment results were normalized to respective controls, which received the value 1. Data are means ± SEM from five independent experiments. *P < 0.05, **P < 0.01 indicate significant difference in comparison with the matching control time point.

We then examined whether PROKs activated adherent macrophages in vitro. To this end, three detection tools were used: 1) ITGB2 mRNA levels in luteal cell/macrophage cocultures, 2) measurement of mononuclear adherent cell number, and 3) TNF mRNA expression.

Both PROKs (50 nM) induced time-dependent increases in ITGB2 mRNA levels in the cocultures of luteal cells and macrophages (Fig. 9A). This elevation became significant at 48 h. The effect of PROK2 was more pronounced than that of PROK1 (a 3-fold increase by PROK2 compared with a 2.5-fold increase by PROK1; Fig. 9A). As indicated by the XTT assay, PROKs (50 nM, 24 h) elevated the number of adherent, metabolically active cells almost to the same magnitude as the E. coli sonicate (15 µg/ml, 24 h; Fig. 9B). Finally, data presented in Figure 9C show that PROKs (50 nM, 24–48 h) stimulated TNF mRNA expression in the macrophage cell fraction, where the two peptides induced approximately a 2-fold increase. These levels were almost half of the maximal stimulation of TNF mRNA induced in macrophages by the E. coli sonicate (4.5-fold increase; data not shown).


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  		FIG. 9. Activation of monocytes by PROKs as evaluated by ITGB2 expression in coculture of luteal cells with monocyte (A), XTT assay (B), and TNF expression by macrophages (C). A) PBMCs were added to luteal cells and incubated with PROKs (50 nM) for up to 48 h. Total RNA was isolated from adherent cells, and ITGB2 mRNA levels were measured by real-time PCR. Within each experiment results were normalized to respective control (1.0). Data are means ± SEM from three independent experiments. Different letters (a, b in instances of PROK1 and A, B in instances of PROK2) indicate significant difference among the different time points (P < 0.05). B) Measure of viable adherent monocytes by XTT assay: monocytes were incubated with PROKs (50 nM) or E. coli sonicate for 24 h. The percentage of viable adherent cells was calculated as described in Materials and Methods. Data are means ± SEM from five separate experiments. Different letters (a, b) show where differences are significant (P < 0.05). C) Monocytes were incubated with PROKs (50 nM) for 24–48 h. Total RNA was isolated and subjected to real-time PCR for determination of TNF mRNA levels. Within each experiment results were normalized to control (1.0). Data are means ± SEM from five separate experiments. *P < 0.05 denotes a significant difference from control.

DISCUSSION

This study describes the expression profile of PROK1 and its two receptor types in follicles and CL throughout the bovine estrous cycle. We showed that PROK1 mRNA was low at early through late luteal stages but sharply increased during luteolysis. Similarly, in human CL the expression of PROK1 was low at early stage [7, 8], upregulated during midluteal phase to late luteal phase, and reached maximal levels at very late luteal phase, including in apoptotic CL [7, 8]. Elevated PROK1 mRNA levels also were detected in the GC layer of atretic follicles. The mRNA of VEGF, a bona fide angiogenic factor, was the mirror image of PROK1: high during early luteal phase and midluteal phase and low during luteolysis or follicular atresia. It is noteworthy that inverse relationships between PROK1 and VEGF also were observed in vitro, both in a human GC line (SVOG) [15] and in bovine luteinized GCs [38].

We have previously shown that PROK1 promoted the proliferation and survival of luteal ECs [9], suggesting its involvement in the angiogenic process of the CL. Therefore, it was unexpected that during the early luteal stage, when angiogenesis takes place and VEGF levels are highest, PROK1 mRNA would be almost undetectable. Interestingly, an intense staining for PROK1 was observed in SMC layers of blood vessels regardless of luteal stage. Hence, SMC may serve as source for angiogenic PROK1 in the developing CL at early/midluteal stages, whereas its increase in atretic CL may reflect activation or influx of a new cell type. Unlike PROK1, VEGF is expressed mainly by GCs and luteal SCs under the control of LH [39, 40] and hypoxia [15, 41, 42]. The different cell sources of these two factors may provide an explanation for their different mode of gene expression regulation. Furthermore, the contrasting profiles of PROK1 and VEGF in bovine and human CL indicate that, contrary to initial hypotheses, the function of these two proteins may not necessarily be overlapping and that PROK1 also is involved in processes leading to CL regression and follicular atresia. These conditions are characterized by sequential induction of increased chemokine expression, leukocyte infiltration, and apoptosis [26, 36, 43, 44].

Interestingly, there was almost a 10-fold higher expression of PROK1 mRNA levels in the GC layer of large healthy follicles relative to the functionally active CL. This may be related to the fact that most healthy bovine and ovine follicles show signs of apoptosis [45, 46]. Nevertheless, it should not be ignored that atresia was accompanied by much higher levels of PROK1 mRNA (20-fold increase in comparison with healthy follicles). This also was observed in the immunostaining of atretic follicles, which was more intense than that of healthy follicles. Nevertheless, a more detailed characterization of PROK1 protein levels in follicles and CL awaits further research.

In the course of follicular atresia and CL involution, chemokine expression and leukocyte infiltration increase [23, 25–28]. These stages are therefore considered proinflammatory events [24, 43, 47, 48]. Monocytes and macrophages are the most abundant immune cells detected in regressed CL, followed by CD8+ and CD4+ T lymphocytes [23, 25, 26, 28]. Our data confirmed that ITGB2, a pan-leukocyte cell marker, was highly expressed in atretic follicles and regressing CL. ITGB2, together with {alpha}-chain of integrin β2, forms active transmembrane molecules mediating firm adhesion to endothelial adhesion molecules and enables leukocytes to leave the blood circulation [49]. Lipopolysaccaride (LPS) is known to stimulate mononuclear phagocytes to synthesize cytokines such as TNF, interleukin 1B (IL1B), and IL6, which play a role in inflammatory reactions and activation of immune responses [50–52]. Therefore, elevated levels of PROK1 in LPS-activated macrophages suggest a role for this peptide in the inflammatory response. This possibility is supported by other reports as well [5, 6]. High levels of PROK1 mRNA were observed in inflammatory tissues in rheumatoid arthritis and Crohn disease [6]. In addition, LeCouter et al. [5] reported that the levels of PROK2 were elevated in infiltrating cells at sites of inflammation, such as the tonsil and appendix.

The positive relationship between PROK1 and ITGB2 curves suggested that PROK1 may be synthesized by leukocytes, possibly macrophages. Indeed, PROK1 immunostaining was identified in macrophages in sections of regressed CL. Direct evidence supporting this proposition was provided by FACS analyses showing that ITGB2-stained cells also were PROK1 positive and that their numbers increased in regressing CL and atretic follicles.

Additionally, we demonstrated here that PROK1 mRNA was elevated in monocytes activated in vitro. While infiltrating leukocytes may account for the major part of elevated PROK1 observed during CL regression, immunohistochemical staining as well as FACS scans suggested the presence of PROK1 in other luteal cell types. It may therefore be assumed that apoptosis of luteal cells would be associated with elevated PROK1 levels. However, there was no increase in PROK1 mRNA expression in luteal cells cultured with proapoptotic factors, such as FASLG and C2-ceramide [36, 37], although they decreased cell viability. The factors or conditions responsible for PROK1 induction in these cells remain, however, to be identified.

Data reported here show that not only is PROK1 expressed by activated monocytes, but that PROK1 further activates these cells. Several lines of evidence support this contention: PROKs increased ITGB2 mRNA expression in a time-dependent manner in cocultures of luteal cells with monocytes, suggesting increased monocyte adherence and/or ITGB2 expression per cell. Both of these responses are indicative of increasing monocyte activation [50, 53, 54]. Furthermore, when cultured alone, PROKs elevated the number of adherent, metabolically active mononuclear cells. Finally, PROKs elevated TNF mRNA levels in macrophages, which provides clear evidence for macrophage activation. Similar findings were reported by Dorsh et al. [6], who had demonstrated that treatment of human peripheral blood monocytes with PROK1 primed the cells to release inflammatory cytokines, including TNF. Another member of the PROK family, PROK2, was reported to induce both migration of monocytes and the secretion of proinflammatory cytokines from macrophages [18]. Collectively, these findings suggest that PROKs may be involved in recruitment and activation of leukocytes. In this context it is interesting to note that another potent monocyte-recruiting peptide, monocyte chemoattractant protein 1 (CCL-2), also is expressed during luteal regression [26, 27].

Mice [18] and bovine (data not shown) PBMCs and macrophages predominantly express mRNA for PROKR1. In addition, PROK2-induced chemotaxis and cytokine modulation were abolished in Prokr1 knockout mice [18], suggesting that the activities exerted by PROKs on macrophages are mediated by PROKR1. The pattern of PROK receptor gene expression in bovine CL and follicles supports this concept: PROKR1 (but not PROKR2) mRNA levels were elevated during luteal and follicular regression.

We previously characterized PROKs as angiogenic factors promoting proliferation and survival of luteal ECs [9]. The present study ascribes an additional role to PROK1 in the bovine ovary; namely, its involvement in enhancing the recruitment and subsequent activation of leukocytes in atretic follicles and regressing CL. The CL is a heterogeneous gland, the cell composition of which varies dynamically during different stages of the cycle [19, 22, 23, 28, 55]. Therefore, it is not surprising that multifunctional molecules, such as PROK1, assume different roles during different cycle stages. The findings reported here also highlight the importance of studying the isolated cell populations in addition to studying the CL as a whole.

Correspondence: 1Rina Meidan, Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. FAX: 972 89 465 763; e-mail: rina.meidan{at}huji.ac.il

Received: 18 June 2006.

First decision: 17 July 2006.

Accepted: 19 December 2006.

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